Development and parameter assessment for vertically aligned platinum wire aptasensor arrays for impedimetric detection of cardiac biomarkers

ABSTRACT

The invention relates to ex-situ biosensors that impedimetrically detect cardiac biomarkers of interest in a bodily fluid sample derived from a patient. The biosensors include a multi-array of vertically aligned platinum wires having immobilized thereon an aptamer that is selected to specifically and selectively bind to the cardiac markers of interest. The biosensors are contacted with a portion of the bodily fluid sample, and the aptamer binds to the cardiac markers of interest in the bodily fluid sample. As a result, an electrochemical impedance signal is generated and therefore, a change in electrochemical impedance is indicative of the presence of the cardiac markers of interest in the bodily fluid sample. The biosensors are point-of-care, on-demand devices that can be used in a medical environment, as well as in domestic settings.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National Stage Application under 35 U.S.C. §371 of International Application No. PCT/2016/055299, filed on Oct. 4,2016, which claims priority under 35 U.S.C. § 119(e) from U.S.Provisional Patent Application No. 62/237,104, filed on Oct. 5, 2015,both of which are entitled “DEVELOPMENT AND PARAMETER ASSESSMENT FORVERTICALLY ALIGNED PLATINUM WIRE APTASENSOR ARRAYS FOR IMPEDIMETRICDETECTION OF CARDIAC BIOMARKERS,” the contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to multi-array, vertically aligned, platinum wireaptasensors for impedimetric detection of cardiac biomarkers in abiological sample, and diagnosis as well as prognosis of cardiovasculardisease, including gauging the risk of cardiovascular disease in apatient. More particularly, the invention relates to an aptasensordevice to provide on-demand, point-of-care screening, analysis andresults.

BACKGROUND

Cardiovascular disease (CVD) is the leading cause of death in developedcountries all around the world, including the United States. CVDencompasses several medical conditions of the heart and blood vessels,including coronary heart disease (CHD), which is the blockage of bloodsupply to the heart by the build-up of fatty deposits in the coronaryarteries. Unattended, CVD can ultimately result in heart failure,stroke, and possible death. Statistics released by the American HeartAssociation indicate that CVD is the leading health problem and cause ofdeath in the United States. Approximately 85.6 million people in thecountry suffer from some form of CVD, and one out of every three deathsresults from heart disease, stroke and other CVDs, claiming more livesthan all forms of cancer combined. In the United States alone, CVDsaccount for 31.9% of all deaths. According to statistics of the WorldHealth Organization, CVD is the leading cause of death globally, andapproximately 17.5 million people died from CVD in 2012. Nearly half thedeaths resulting from CVD are the result of coronary heart diseasealone. Direct and indirect costs of CVDs amount to more than $320.1billion, constituting nearly 17% of national health expenditures.Unfortunately, the prevalence and costs of CVDs are projected tocontinue to increase over the years. It is estimated that the prevalenceof CVD will rise from 37.8% to 40.5% and the costs will escalate from$350 billion to $818 billion by 2030.

Despite the considerable advances in the field of medicine, especiallyin CVD treatment, CVDs are and will continue to remain the leading causeof death in developed countries. This statistic is, at least in part,the result of a lack of standard method for diagnosis of CVD. Thedisease is clinically silent until serious complications arise.Diagnosis of CVD typically follows the onset of chest pains,electrocardiography results, and biochemical marker testing from bloodsamples. There are disadvantages associated with these diagnosissymptoms. Chest pains are not associated with CVDs alone;electrocardiography results are not always reliable; and processingblood sample paperwork and lab work can involve a period of severaldays, allowing critical time to lapse. Furthermore, imaging techniquessuch as magnetic resonance imaging (MRI), ultrafast computerizedtomography (CT), and coronary and cerebrovascular angiography utilizeexpensive laboratory equipment and involve invasive procedures.Electrocardiography (ECG), based on electrical charges that occur duringthe heart cycle, is commonly used as a diagnostic tool for CVD due toits affordability and availability. However, ECGs produce a staticpicture that is not always indicative of the severity of the underlyingCVD conditions and has only a 50% sensitivity. Thus, the lack ofstandard diagnostic methods and slow turnover for processing bloodsamples in hospital laboratories indicate the critical necessity of apoint-of-care diagnostic biosensor for the rapid and sensitive detectionof cardiac markers in blood.

Antibodies and enzymes are commonly utilized detection elements inbiosensors due to their high affinity and specificity. However, thereare disadvantages associated therewith—namely, antibodies and enzymeshave relatively short shelf lives, are restricted by in vivo parameters,have batch-to-batch variation, and are sensitive to chemical ortemperature changes. The use of synthetic aptamers (as compared tobiological antibodies and enzymes) provide significant improvements fora biosensor. Aptamers are synthetic oligonucleotide sequences that aresynthesized to bind to their target with high affinity and specificity,and therefore provide significant improvements for a biosensor. Thesynthetic process of producing aptamers ensures the following propertiesand characteristics: high stability in various environments, long shelflives, and minimal batch-to-batch variation, while maintaining theiraffinity and specificity. The aptamer-based biosensor can provide astable system that may be more easily translated to a medical device.Furthermore, the small size and lack of hydrophobic core in aptamers canprevent aggregation, which has been found to be problematic inantibodies.

Two particular biomarkers of interest in diagnosing the risk andprevalence of CVD are brain natriuretic peptide (BNP) and Troponin-T(TnT). BNP is a polypeptide secreted by the ventricles of the heart intothe bloodstream upon excessive stretching of cardiomyocytes and istherefore an indicator of cardiac stress. TnT is a protein released intothe bloodstream upon myocyte injury or death and is therefore anindicator of cardiac injury. Tailoring a biosensor to detect these twocardiac markers is of particular interest to the cardiovascular healthcommunity.

Synthesis of an impedimetric device requires a conductive materialinterface. There are advantages associated with the use and selection ofplatinum as the material interface, such as its chemically andelectrochemically inert noble metal status, high conductivity, andbiocompatibility. However, there are also disadvantages associated withplatinum—most biosensor studies conducted on platinum interfaces stillutilize antibodies and enzymes as detection elements, and often useplatinum electrodes or nanoparticles in tandem with other materialinterfaces, such as, carbon nanotubes/nanocomposites, graphene,chitosan, silica, polymers, or gold. It is not known in the art todevelop an aptamer-based biosensor on a platinum interface alone.

Various existing biosensor-based technologies are primarily based onfluorescent-immunoassays that require fluorescently-labeled antibodiesand a bench-top analyzer for the fluorescent assay. Common immunoassaysinclude membrane-based immunoassays such as lateral flow devices (LFD)and enzyme-linked immunosorbent assays (ELISAs). These tests are highlydependent on the use of fluorescently labeled antibodies andspectrophotometers for analysis of fluorescence levels. The assays aretypically conducted in laboratories and require significantpre-analytical time and analytical time, which increases turn-aroundtime. In addition, many of these devices require a greater volume ofblood than a typical glucose detector.

In general, there is a lack of standard diagnostic methods, turnover ofprocessing blood samples in hospitals and laboratories is frequentlyslow, and common diagnostic methods are expensive, time-consuming,invasive, requiring the patient to be tested in a medical facility andrequiring the results to be obtained by skilled and trained personnel,which do not promote routine testing. Thus, there is a need for thedevelopment of improved impedimetric aptasensors capable of providingone or more of efficient, early, convenient (e.g., point-of-care,on-demand), inexpensive, rapid, minimally or non-invasive and accurateimpedimetric detection and screening of multiple cardiac markerssimultaneously and a diagnosis or prognosis of CVD risk in patients, inorder to provide preventative medication and therapeutic treatment forfavorable outcomes, which may be utilized outside the confines of ahospital or other medical facility, e.g., in a domestic setting, suchas, a patient's home.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a portable, ex-situ system toimpedimetrically detect cardiac biomarkers of interest in a patient. Thesystem includes a conductive material interface having a surface, andincluding an epoxy substrate and a multi-array of vertically alignedplatinum wires cast in the epoxy substrate, a biological sensor agentapplied to the surface of the conductive material interface. Thebiological sensor agent includes an immobilization agent, and at leastone aptamer selected to interact with the immobilization agent andselected to bind with the cardiac biomarkers of interest. The systemalso includes a signaling agent consisting of an electrochemicalimpedance signal generated by binding of the aptamer with the cardiacbiomarkers of interest; and a bodily fluid sample derived from thepatient and in contact with the aptamer. A change in electrochemicalimpedance is indicative of a presence of the cardiac biomarkers ofinterest in the bodily fluid sample, and an absence of a change inelectrochemical impedance is indicative of an absence of the cardiacbiomarkers of interest in the bodily fluid sample.

An end of the platinum wires serves as the point of contact for theelectrochemical impedance signals to be transduced, allowing for aninterpretable reading of the output.

The bodily fluid sample can be a blood sample. The cardiac biomarkerscan be selected from C-reactive protein, Creatinine Kinase, TroponinT,Myoglobin, IL-6, IL-18, Brain Natriuretic Peptide, and D-Dimer.

The aptamer can be conjugated with biotin. The immobilization agent canbe selected from the group consisting of avidin, streptavidin,neutravidin and mixtures thereof. The immobilization agent can beapplied to a treating agent, and the treating agent can be applied tothe surface of the conductive material interface including themulti-array of vertically aligned platinum wires.

The multi-array of vertically aligned platinum wires can be arranged onthe surface of the substrate in a circular configuration.

The aptamer can be effective to impedimetrically detect simultaneously aplurality of cardiac biomarkers in the bodily fluid sample.

In another aspect, the invention includes a method of detecting cardiacbiomarkers in a bodily fluid sample of a patient. The method includesobtaining the bodily fluid sample from the patient; forming a detectiondevice including forming a conductive material interface having asurface, providing an epoxy substrate, obtaining a multi-array ofvertically aligned platinum wires, and casting the multi-array ofvertically aligned platinum wires in the epoxy substrate; polishing thesurface of the conductive material interface; forming a biologicalsensor agent including applying an immobilization agent to the surfaceof the conductive material interface, selecting an aptamer toselectively bind with the cardiac biomarkers of interest, andinteracting the aptamer with the immobilization agent; contacting theaptamer with the bodily fluid sample; generating an electrochemicalimpedance signal as a result of the aptamer binding with the cardiacbiomarkers of interest; and assessing a presence or an absence of achange in electrochemical impedance. The presence of a change inelectrochemical impedance is indicative of the presence of the cardiacbiomarkers of interest in the bodily fluid sample, and the absence of achange in electrochemical impedance is indicative of the absence of thecardiac biomarkers of interest in the bodily fluid sample.

The electrochemical impedance signal can be transduced to aninterpretable read-out value. The electrochemical impedance signal canbe connected to a hand-held device that is effective to display theread-out value.

The detection device can be in the form of a test strip and the method,can include contacting the bodily fluid sample with the test strip;assessing a visual change to the test strip; correlating the visualchange with a chart or key; and based on said correlating, determiningif the visual change is indicative of the presence of a change inelectrochemical impedance and the presence of the cardiac biomarkers inthe bodily fluid sample. The visual change can also be a color change.

The polishing of the surface of the conductive material interface can beconducted to provide a surface roughness in a range from about 320 gritto about 2400 grit.

BRIEF DESCRIPTION OF THE DRAWINGS

A further understanding of the invention can be gained from thefollowing description of the preferred embodiments when read inconjunction with the accompanying drawings in which:

FIG. 1 shows cyclic voltammagrams and Nyquist interpretations ofElectrochemical Impedance Spectroscopy (EIS) experiments conducted withAg wire reference electrode and Pt wire counter electrode for platinumelectrodes of different platinum wire diameters subjected to threepolishing grits, in accordance with certain embodiments of theinvention;

FIG. 2 shows Nyquist interpretations of EIS experiments conducted in 5mM (Fe(CN₆)^(3-/4-)) in 10 mM PBS, with 5 μm polished 0.5 mm diameterplatinum working electrodes, Ag wire reference electrode, and Pt wirecounter electrode, in accordance with certain embodiments of theinvention; and

FIG. 3 shows calibration curves depicting average percent change incharge-transfer resistance as a function of concentration and standarderror for platinum-based aptasensors of three polishing grits atdifferent platinum wire diameters, in accordance with certainembodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to multi-array, impedimetric, vertically alignedplatinum aptasensors for the impedimetric detection and screening of atleast one cardiac biomarker, or multiple cardiac biomarkerssimultaneously, present in bodily fluid, such as, blood, that signal theonset of, for example, artherosclerosis and/or inflammation of cardiactissue and/or myocardial infarctions and/or cardiac dysfunctions. Moreparticularly, the invention includes an electrochemical assay forelectrochemically detecting cardiac biomarker concentrations that arepresent in a minimal amount of blood, e.g., a few drops of blood, bymeasuring, e.g., quantitatively, impedance changes that occur upon thebinding of antigens to the aptasensors. Further, the invention includesmethods of synthesizing the aptasensors that specifically andselectively bind to an intended target analyte, and employing thesynthesized aptasensors for detecting and diagnosing myocyte stress andinjury in a patient.

The aptasensors are in-vitro (ex-situ) devices that utilize the bodilyfluid sample derived from the patient for impedimetric detection of theone or more cardiac biomarkers. The impedance changes are measured usingelectrochemical impedance spectroscopy (EIS), which is a highlysensitive, label-free technique that allows for changes inelectrochemical impedance resulting from the binding of the aptamer tothe antigen to be transduced into an interpretable read-out value. Thus,the aptasensor electrochemically detects cardiac biomarkerconcentrations present in the minimal amount of blood by measuring theensuing impedance changes occurring upon antigens binding to theaptasensor.

As used in the specification and in the claims, the singular form of“a”, “an”, and “the” may include plural referents unless the contextclearly dictates otherwise.

In general, according to the invention, aptamers are a form ofbiological detection (sensing) agent that are utilized to detect whetherthere exists certain analytes/biomarkers within a subject fluid sample.The term “aptamer” as used herein, refers to an oligonucleotide oroligonucleotide chain that has a specific and selective binding affinityfor an intended target compound or molecule (e.g., analyte) of interest,and is capable of forming a complex with the intended target compound ormolecule of interest. The complexation is target-specific in the sensethat other materials which may accompany the target, do not complex tothe aptamer. It is recognized that complexation and affinity are amatter of degree; however, in this context, “target-specific” means thatthe aptamer binds to the target with a much higher degree of affinitythan it binds to contaminating materials. As used herein, the term“binding” refers to an interaction or complexation between the targetcompound or molecule of interest and the aptamer. Aptamers can be usedin diagnosis by employing them in specific binding assays for the targetcompound or molecule of interest.

As used herein, “biomarkers” refer to naturally occurring or syntheticcompounds, which are a marker of a disease state or of a normal orpathologic process that occurs in an organism. The term “analyte,” asused herein, refers to any substance, including chemical and biologicalagents that can be measured in an analytical procedure. The term “bodilyfluid”, as used herein, refers to a mixture of molecules obtained from apatient. Bodily fluids include, but are not limited to, exhaled breath,whole blood, blood plasma, urine, semen, saliva, lymph fluid, meningealfluid, amniotic fluid, glandular fluid, sputum, feces, sweat, mucous andcerebrospinal fluid. Bodily fluid also includes experimentally separatedfractions of all of the preceding solutions or mixtures containinghomogenized solid material, such as tissues and biopsy samples.According to the invention, biomarkers and/or analytes are detectable inbodily fluid, such as, but not limited to, a minute volume of blood.

An “array” is an intentionally created collection of molecules. Themolecules in the array can be identical or different from each other.

The systems (e.g., biosensors) and methods of the invention can includeat least one biological sensor agent and at least one signaling agentwherein the biological sensor agent(s) and signaling agent(s) togetherprovide a means for detecting, signaling, and/or quantifying targetcompounds of interest in bodily fluids, such as, blood. The biologicalsensor agent is selected for its ability to specifically and selectivelyinteract with and bind to (only) the target analyte/biomarker molecules.In accordance with the invention, the biological sensor agent isattached to the surface of a conductive material interface. Thebiological sensor agent can be introduced by functionalization of thesurface of a conductive material interface. The biological sensor agentcan be directly attached to the conductive material interface orindirectly attached by employing linker molecules, such as, but notlimited to, proteins. The conductive material is a multi-array ofvertically aligned platinum wires cast in an epoxy substrate. Thebiological sensor agent is in the form of an aptamer. For example, anaptamer-linked protein can be immobilized on a surface of the conductivematerial interface. The aptamer can be conjugated to a signaling agent,e.g., the electrochemical impedance signal. The signaling agent isdetectable under preselected conditions, e.g., after aptamer binding tothe analyte/biomarker of interest. In accordance with the invention,signaling is related to a change in impedance, upon binding of theaptamer with the analyte/biomarker of interest. An end of the platinumwires provides a point of contact for an electrochemical impedancesignal to be transduced to an interpretable read-out value.

In certain embodiments, the invention utilizes platinum wire as aconductive material interface and platform for a biosensing surface. Theimmobilized biological sensor is applied to the platform. Theimmobilized biological sensor includes the aptamer, e.g., biotinylatedaptamer, and the immobilization agent. Furthermore, the signaling agentincludes the electrochemical impedance signal. The aptasensor istailored to detect various cardiac markers predictive of cardiovasculardisease (CVD) in bodily fluids, primarily, but not limited to, blood, todetermine the risk state of a patient for CVD. The sample of bodilyfluid can be a minute volume, such as, for example, a few drops (e.g.,about 1-5 drops) of blood, and the determination can be obtained in arelatively short period of time, such as, for example, about severalminutes to five minutes. The cardiac markers can include C-reactiveprotein, Creatinine Kinase, TroponinT, Myoglobin, IL-6, IL-18, Brainnatriuretic Peptide, and D-Dimer. Brain Natriuretic peptide (BNP) is anindicator of myocyte stress and Troponin-T (TnT) is an indicator ofmyocyte injury. In contrast, known systems for detecting CVD includemagnetic resonance imaging (MRI), computerized tomography (CT),electrocardiography (ECG) and invasive techniques, such as, coronary andcerebrovascular angiography. For systems focused on blood work,enzyme-linked immunosorbent assays (ELISA) and lateral flow devices(LFD) can be employed. These analyses and devices for detection involveexpensive equipment, highly-skilled and trained personnel for properanalysis, associated risks and a significant amount of time forprocessing, which includes analytical time, e.g., the duration of theassay, and pre-analytical time, involving paperwork, drawing samples,labeling samples and enormous preparation time. The multi-arrayaptasensors in accordance with the invention provide portable,point-of-care, on-demand devices that can be utilized in the absence ofexpensive equipment and highly trained professionals to assess levels ofcardiac markers in the blood at the patient's bedside, for example,within a short period of time, such as, several minutes. Since the useof these aptasensors do not require much skill or training, theyrepresent a simple and facile mode of detection.

Further, known impedimetric devices utilize antibodies and enzymes asdetection elements, and platinum is used in tandem with other materialinterfaces such as carbon nanotubes/nanocomposites, graphene, chitosan,silica, polymers, or gold. In contrast, the multi-array aptasensors inaccordance with the invention utilize platinum alone as the materialinterface.

Synthesis of the multi-array aptasensors, in accordance with theinvention, includes the use of appropriate linkers and proteins toimmobilize cardiac marker-specific aptamers to the surface of theplatinum wires. The platinum wire arrays, e.g., vertically aligned, areembedded in an epoxy mold, e.g., in a circular fashion or pattern, andthe surface of the epoxy mold is polished, e.g., to approximately 50 nm,for surface exposure. The surface then can be treated with a thiol-basedcompound, such as, an aminothiol, including but not limited to,cysteamine and/or glutaraldehyde. An immobilization agent, such as,avidin, is adsorbed thereon. One or more aptamers is conjugated withbiotin. The biotinylated aptamers for the above-described cardiacmarkers interact with the immobilization agent to develop the biosensingsurface. Application of the treating agent and the immobilization agent,and interaction of the biotinylated aptamers can be carried out in asequential manner, to develop the biosensing surface. As previouslydescribed, aptamers are similar to antibodies in that aptamers areoligonucleotide sequences that are highly specific for their designatedantigen. However, unlike antibodies, aptamers can undergo denaturationand renaturation. The aptasensors can therefore be regenerated in thepresence of certain solvents, thus providing a reusable and regenerativesensor for potentially continuous use rather than one-time detection (asin commercially known glucose sensors). Thus, aptamers are more robustwith a longer shelf-life and more importantly, allowing for aptasensorsto be reusable rather than only a one-time, single-use assay.

An electrochemical sensor can be used to measure a change in output of asensing element caused by chemical interaction of a target marker on asensing element. In accordance with the invention, electrochemicalimpedance spectroscopy (EIS) is the technique, e.g., sensor agent,utilized to characterize the surface of an aptasensor at various stagesof development. EIS is a highly sensitive and label-free technique thatallows for changes in electrochemical impedance resulting from thebinding of the aptamer to the antigen. The electrochemical impedance canbe transduced to a read-out value. Thus, the aptasensor is capable ofelectrochemically detecting cardiac biomarker concentrations that arepresent in a minimal amount of blood by measuring the impedance changesthat occur upon the binding of antigens to the aptasensor. Theimpedimetric detection of the cardiac biomarker can be performed withinminutes, and the aptasensor can be reused for this purpose multipletimes.

In accordance with the invention, vertically aligned modified platinumwire-based aptasensors are provided for the impedimetric detection ofcardiac markers. The aptasensors are synthesized by casting uprightplatinum wires in epoxy. The wires can be cast in various configurationsand patterns. In certain embodiments, the wires are cast in a circularpattern. The diameter of the wires may vary and can range from about0.25 mm to about 1.0 mm. In certain embodiments, the diameter is about0.25 mm or about 0.5 mm or about 1.0 mm.

One end of the wire is cast in the epoxy and the opposite end has animmobilized aptamer attached thereto. Thus, the wires are utilized forfunctionalization and establishing electrical connection.

The resulting platinum electrodes are polished using polishing media,such as, but not limited to, silicon carbide (SiC), to various differentgrits and functionalized to bind the cardiac biomarker-specific aptamersto the surface. The surface roughness can vary and, for example, thepolishing grit size, can range from about 320 grit (e.g., about 50 μm)to about 2400 grit (e.g., about 50 nm). In certain embodiments, the gritsize is about 320 grit or about 1200 grit (e.g., about 5 μm) or about2400 grit. It is contemplated and understood that the impedimetricdevices can be tested against various clinically relevant concentrationsof cardiac biomarker to determine the ideal wire diameter and polishinggrit.

In certain embodiments of the invention, the aptasensors utilize 0.5mm-diameter wires polished to about 1200 grit (e.g., 5 μm) size.

Electrochemical impedance spectroscopy (EIS) can be employed as a modeof impedimetric detection for the one or more cardiac biomarkers.

Impedimetric biosensors provide one or more of the following featuresand advantages as compared with known biosensors of CVD: highlysensitive, low cost, allow for rapid analysis and miniaturization, andlabel-free, thus significantly reducing the complexity of biosensordevelopment.

There are various conventional mechanisms for functionalizing, e.g.,attaching an aptamer thereto, the platinum wires including, but notlimited to, adsorbing a binding material thereon. The binding materialis selected based on its capability to bind particular aptamer.Non-limiting examples of suitable binder materials include avidin,streptavidin, and neutravidin. In certain embodiments, neutravidin ispreferred. Further, the aptamer for binding to the avidin is selectedbased on its capability to interact with the target biomarker.Non-limiting examples of suitable aptamer include biotinylated aptamerselected specifically for cardiac biomarkers, such as, but not limitedto, those described herein, for example, BNP and TnT. Thus, the avidinis immobilized on the surface of the platinum wires and the biotinylatedaptamer attaches to the avidin.

The process of biotinylation generally includes covalently attachingbiotin to a protein, nuclei acid or other molecule. Biotin is known tobind to avidin with high affinity. The aptamer can be biotinylatedchemically or enzymatically using conventional processes and apparatus.

In certain embodiments, the multi-array of platinum wires is embedded inan epoxy substrate, the surface of the epoxy substrate is polished andthe wires on the surface of the epoxy substrate are treated with avidinfollowed by biotinylated aptamer. The biotinylated aptamer can includebiotinylated proteins. In certain embodiments, the biotinylated aptameris selected based on its ability to interact with cardiac biomarkers.Thus, BNP and TnT aptamer may be selected to interact with BNP and TnTbiomarker, respectively. These cardiac biomarkers are released intobodily fluids, e.g., blood, for example, as a result of myocytestretching or injury or death.

The biosensors developed in accordance with the invention may functionas ex-situ biosensors. A portable (e.g., point-of-care, on-demand)device, such as, a handheld device, may be developed. There are variousmechanisms that are known in the art to produce a handheld device thatmay be employed with the biosensors, and are suitable for use with thebiosensors of the invention. In certain embodiments, the electrochemicalimpedance signal is transduced to a read-out value, and the read-outvalue is displayed on a handheld device. The handheld device can be anelectronic device. Alternatively, the handheld device can include, forexample, a test strip similar to conventional glucose sensors which areknown in the art. There is typically a corresponding standard chart orkey used to interpret the results displayed on the test strip. In theseembodiments, the test strip is contacted with a patient bodily fluidsample, such as by applying the sample, e.g., a few drops, to the teststrip or by dipping/immersing the test strip into the bodily fluidsample. The test strip is then visually observed or inspected todetermine whether there is a visible change, such as a change in color,based on its contact with the sample. The mere presence of a visualchange, such as color change, is indicative of a change ofelectrochemical impedance, e.g., binding of the aptamer in the teststrip with the cardiac biomarkers in the bodily fluid sample, andtherefore, the presence in the sample of the cardiac biomarkers ofinterest. Further, the corresponding key or chart can include varyingdegrees or intensity of change. The degree or intensity of visual changeon the test strip is correlated to a particular quantitative amount ofthe electrochemical change and corresponding level of cardiac biomarkersof interest in the sample. Similarly, the absence of a visual change onthe test strip is indicative of the absence of the cardiac biomarkers ofinterest in the patient bodily fluid sample.

For example, in accordance with certain embodiments of the invention, abodily fluid sample, such as blood, is obtained or removed from apatient. Further, the sample can be obtained or removed by the patient.At least a portion of the sample is deposited on the test strip andwithin a time period, e.g., seconds or a few minutes, a change in colorof at least a portion of the test strip is visually observed based onthe cardiac biomarkers in the sample interacting with the test strip,e.g., biosensor. The particular color and/or the intensity of the colorchange is compared and matched with a key to determine the level of thecardiac biomarker, e.g., BNP and/or TnT, in the sample. Based on thevisible change of the biosensor, the presence or absence or particularconcentration of the cardiac biomarker is determined efficiently andaccurately. The response time may be in minutes or even seconds, and theresults can be obtained by the patient in a domestic setting, withoutthe need for medical personnel, laboratory equipment and a medicalfacility.

Therefore, impedimetric biosensors in accordance with the invention areideal portable, e.g., point-of-care, on-demand, diagnostics that can beused, for example, at bedside, in ambulances, or even during clinicalvisits as a useful screening device for the detection of cardiacbiomarkers and therefore, the diagnosis of CVD.

Further, in accordance with the invention, impedimetric biosensorsexhibiting the following attributes are provided: (i) re-usableaptamer-based electrochemical assay; (ii) multiple cardiac biomarkerdetection in a single setting; and (iii) amenable to hand-held modeltranslation.

Point-of-care handheld aptasensors in accordance with the inventionallow patients to frequently detect and measure their cardiac biomarkersand therefore, assess their individual CVD risk and to monitor howdifferent lifestyle changes can reduce this risk. In addition, theaptasensors are inexpensive, e.g., comparable in price to blood-basedglucose biosensors that are currently commercially available. Further,existing insurance codes for glucose biosensors and cardiac biomarkertesting could be readily applied to aptasensors for full or partialreimbursement of the cost. Thus, patients can affordably, routinely andrapidly detect and measure their cardiac biomarkers.

In an emergency room setting, significant minimization of turn-aroundtime may be realized, resulting in more efficient allocation ofresources and providing more effective care for patients. For example,decreasing the time of diagnosis can reduce the time required to make anadmission decision and therefore, ensure administration of rapid care tothe patient. In addition, decreasing the time of diagnosis may alsoensure that patients suffering from less severe conditions are notallocated more expensive, redundant resources.

In certain embodiments, the aptasensors can be tailored with a wirelesschip to allow for wireless transmission of biomarker levels to apatient's electronic health records. Thus, reducing the amount ofpaperwork necessary and allowing the physician to directly view trendsin the levels of biomarkers and detecting early a precarious patient CVDsituation. In an overall health-care system setting, the aptamers mayeventually allow for the replacement of antibodies with aptamers forimmunoassays.

It should be understood that the embodiments described herein and theexamples provided below are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application.

EXAMPLES

In accordance with the invention, impedimetric vertically-alignedplatinum wire-based aptasensors for the detection of cardiac markers BNPand TnT were developed and parameter assessment (specifically, diameterof wire and surface polishing) was conducted. Upright platinum wires ofvarying diameters were cast in epoxy such that one end was utilized forfunctionalization and the other end was used to establish electricalconnection. The resulting platinum electrodes were accordingly polishedto various different grits, functionalized to bind the BNP andTnT-specific aptamers to the surface, and tested against variousclinically relevant concentrations of BNP and TnT to determine the idealparameters (wire diameter and polishing grit) for biosensor and medicaldevice development.

1. Example

Three surface polishes (50 μm, 5 μm, 50 nm) and three platinum wirediameters (0.25 mm, 0.5 mm, and 1.0 mm) were characterized,functionalized, and then tested against clinically relevantconcentrations of BNP and TnT to assess which parameter was optimum fordetecting BNP and TnT without losing precision or sensitivity. The idealparameter for the vertically-aligned platinum wire array was found to be0.5 mm diameter wire polished to 5 μm. Therefore, the feasibility of thedeveloped platinum aptasensor and the ideal parameters required for theaptasensor were demonstrated.

2. Experimental Procedure

2.1. Reagents

Potassium ferrocyanide and potassium ferricyanide were purchased fromFisher Scientific; cysteamine was purchased from Acros Organics;glutaraldehyde was purchased from Sigma-Aldrich; avidin was purchasedfrom Thermo-Fisher Scientific;

brain natriuretic peptide (BNP) and TroponinT (TnT) biotinylatedaptamers were purchased from OTC Biotech; brain natriuretic peptideantigen was purchased from ABDSerotec; and TroponinT antigen waspurchased from LeeBio. All the aqueous solutions were either prepared inPhosphate-Buffered Saline (PBS) purchased from Lonza or in Milliporede-ionized water (18 M Ωcm⁻¹).

2.2. Electrode Preparation

Vertically aligned platinum wires (0.25 mm dia, 0.5 mm dia, and 1.0 mmdia, 99.9% metals basis, Alfa Aesar) were cast in a non-conducting epoxyresin disk (Buehler) in a circular pattern and polished to 50 μm on 320grit, 5 μm on 1200, and 50 nm on 2400 grit silicon carbide paper (AlliedHigh Tech Products, Inc.). The resulting disk was sonicated inde-ionized water followed by 95% EtOH, for five minutes each prior toelectrochemical characterization and functionalization.

2.3. Electrochemical Characterization

All electrochemical characterization was carried out using the Gamryseries G Potentiostat in an electrolyte solution of 5 mM potassiumferro/ferricyanide redox couple in 10 mM PBS (Fe(CN₆)^(3-/4-)) withsilver wire as the reference electrode and platinum wire as the counterelectrode. For electrode characterization, both cyclic voltammetry (CV)and electrochemical impedance spectroscopy (EIS) experiments wereconducted, and for functionalization and antigen binding assessments,the EIS experiments were conducted after each step. CV experiments werecarried out across a potential range of −0.4V to 0.6V at a scan rate of100 mV/s, and EIS experiments were carried out across a frequency rangeof 300,000 Hz-0.01 Hz with an AC amplitude voltage of 10 mV rms.Resultant Nyquist plots were analyzed using Z-view (Scribner Associates,Inc.) to determine the charge-transfer resistance values.

2.4. Electrode Functionalization

The platinum electrodes were treated with 10 mg/mL cysteamine preparedin de-ionized water for 1 hour at room temperature, followed by 25%glutaraldehyde in water for 1 hour at room temperature for thiolationand carboxylation of the surface. The surface was then treated with 1mg/mL neutravidin prepared in 10 mM PBS for 2 hours at room temperature,followed by incubation with biotinylated aptamer (for 2 hours at roomtemperature). The electrodes were then stored in PBS at 4° C. until timeof use.

2.5. Antigen Testing

Four concentrations for both BNP and TnT were prepared, each within theclinical range for low to high risk for cardiovascular disease.BNP-aptamer biosensors were successively treated with 0.2 ng/mL, 0.6ng/mL, 1.0 ng/mL, and 2.0 ng/mL BNP, and TnT-aptamer biosensors weresuccessively treated with 0.005 ng/mL, 0.01 ng/mL, 0.02 ng/mL, and 0.04ng/mL TnT, to develop a calibration curve for future biosensor testing.EIS measurements were taken after each antigen incubation for thedevelopment of the calibration curves.

3. Results & Discussion

3.1 Characterization of Electrode Parameters

Three disks of vertically aligned platinum wire electrodes were prepared0.25 mm diameter, 0.5 mm diameter, and 1.0 mm diameter platinum wireelectrodes and each disk was polished to grits of 50 μm, 5 μm, 50 nm.Thus, a total of nine parameters were characterized, functionalized, andtested for antigen detection. Electrochemical characterization of thebare platinum electrodes for the nine possible parameters (as shown inFIG. 1) demonstrated that an increase in diameter led to an increase incurrent passage through the electrode (as shown in FIG. 1, views a-c)and accordingly, a decrease in the charge-transfer resistance (as shownin FIG. 1, views d-f). As the diameter of the electrode increased, thepeak currents had a tendency to cluster together, demonstrating that thepolishing had less of an impact on the current passage. However, the 50μm polished electrode consistently had the highest peak currents, whilethe 5 μm consistently had the lowest peak currents. The 50 μm polishedelectrodes had the lowest charge-transfer resistance and the 5 μmpolished electrodes had the highest charge-transfer resistance. Whilethe range of charge-transfer resistances across polishing decreased,there were substantial differences between polishing, especially between50 μm and 5 μm. This established EIS as a highly sensitive technique ascompared to known techniques, such as, cyclic voltammetry.

3.2 Biosensor Functionalization and Antigen Detection

All charge-transfer resistances obtained from EIS characterization atany stage of the experiment fit the equivalent circuit shown in FIG. 2a, which depicted a solution resistance (due to the ferro/ferricyanideelectrolyte) in series with two constant phase element (CPE) components,with each CPE component in parallel to a resistance component. The CPEcomponents were the result of an electrochemical double layer, which wasindicative of exposure of the electrode to the electrolyte, thuscreating two parallel layers of charge the first layer being surfacecharge resulting from the chemical interactions on the surface, and thesecond layer being ions attracted to but loosely associated with thesurface charge (known as a diffuse layer). Each CPE-R_(ct) circuit wasmarked as inner layer or outer layer, with the inner layer being thesemicircular portion of the Nyquist plot, and the outer layer being thesecond portion of the Nyquist plot, which would represent a secondsemicircle with further extrapolation of the plot. The inner layerrepresented the chemical interaction of interest, while the outer layerrepresented the possible interaction of ions with other charged layersof the electrode/biosensor. FIG. 2b demonstrates the multiple chemicalinteractions required to bind the sensor element (aptamers) to theplatinum electrode surface. Cysteamine, glutaraldehyde, avidin, andaptamer were added in succession of one another, followed by storage inPBS (which served as the buffer fluid and as the 0.00 ng/mL baseline forthe antigen detection experiments). As each component was added to thebiosensing surface, the charge transfer increased (FIG. 2b ), withAvidin binding demonstrating the largest change in charge-transferresistance from the previous layer due to its large size compared tocysteamine, glutaraldehyde, and aptamers. Once the biosensors wereprepared, the biosensors were tested for antigen detection for fourclinically relevant concentrations of BNP (FIG. 2c ) and TnT (FIG. 2d )respectively. As the antigen concentration increased, the chargetransfer resistance increased, thus indicating that as more antigenbound to the aptamer on the surface of the biosensor, the impedanceincreased, thus allowing the concentration to be electrochemicallyquantified as a charge-transfer resistance value.

3.3 Linearity and Reproducibility of Calibration Curves for BiosensorParameters

Once all nine parameters were electrochemically tested for both BNP andTnT antigen detection through EIS, the percent change between eachantigen charge-transfer resistance value and the baselinecharge-transfer resistance value was calculated and plotted against theconcentration to determine the calibration curve for each parameter forboth BNP (FIG. 3a-c ) and TnT (FIG. 3d-f ). Saturation in theseclinically relevant levels was indicative of the biosensor's lack ofsensitivity at these crucial concentrations. However, linearity wasindicative of the biosensor's success to detect within the crucialconcentration range and possibly beyond that range as well. Standarderror (n=3) was calculated for each concentration at each parameter,with a smaller standard error being indicative of precision andreproducibility while larger standard errors were indicative ofinconsistency across the electrodes. Therefore, the parameter thatdemonstrated excellent linearity and precision for both BNP and TnTwould be the ideal parameter. At the 0.25 mm diameter, all but oneparameter (50 nm) saturates for BNP (FIG. 3a ), and all parameterssaturate for TnT (FIG. 3d ). At the 0.5 mm diameter, both 50 μm and 5 μmfor BNP demonstrated linearity, but the correlation betweenconcentration and percent change in charge-transfer resistance wassmaller for 50 μm (R²=0.89) than for 5 μm (R²=0.98). All parameters savefor 5 μm saturated in TnT (FIG. 3e ), and the 5 μm calibration curve hadan excellent correlation (R²=0.98). At the 1.0 mm diameter, allparameters saturated for both BNP (FIG. 3c ) and TnT (FIG. 3f ).Therefore, the ideal parameter was determined to be 0.5 mm wire polishedto 5 μm. This parameter may have been ideal due to the fact that roughersurfaces (50 μm) tend to be better for protein attachment than smoothersurfaces (50 nm) due to greater surface area, but rougher surfaces canalso induce greater protein denaturation (Rechendorff 2006,Dolatshahi-Pirouz 2008). Thus, a surface that is between these twospectrum would be ideal (5 μm). The same compromise could be extended towire diameters, where the smaller diameter (0.25 mm) had reduced areafor binding, while the larger diameter (1.0 mm) had larger area forbinding, but also a higher probability of expressing inconsistencies ordefects on the surface (Nishida 1992, Van Noort 2013). Thus, the middlediameter (0.5 mm) was ideal.

4. Conclusions

In summary, a simplistic upright platinum wire-based multi-arrayimpedimetric biosensor was effective to detect markers indicative ofmyocyte stress (BNP) and myocyte injury (TnT). This simplistic designrequired no labeling, was cost-effective, and upon subsequently beingscaled down and miniaturized, requires no expensive and esotericinstrumentation. The ideal parameter of 0.5 mm platinum wire polished to5 μm was determined for achieving effective and consistent detection.

The invention claimed is:
 1. A portable, ex-situ system toimpedimetrically, simultaneously detect multiple cardiac biomarkers ofinterest in a patient, comprising: a multi-array aptasensor, comprising:a conductive material interface, comprising: an epoxy substrate; and amulti-array of vertically aligned platinum wires cast in the epoxysubstrate; a biological sensor agent applied to the multi-array ofvertically aligned platinum wires, the biological sensor agentcomprising: an immobilization agent; and one or more aptamers selectedto interact with the immobilization agent and selected to bind with themultiple cardiac biomarkers of interest; a signaling agent comprising anelectrochemical impedance signal generated by binding of the multi-arrayaptasensor with the multiple cardiac biomarkers of interest; and abodily fluid sample derived from the patient and in contact with themulti-array aptasensor to simultaneously detect the multiple cardiacbiomarkers of interest, wherein a change in the electrochemicalimpedance is indicative of a presence of the multiple cardiac biomarkersof interest in the bodily fluid sample and binding of the multi-arrayaptasensor with the multiple cardiac biomarkers in the bodily fluidsample, and wherein an absence of a change in the electrochemicalimpedance is indicative of an absence of the multiple cardiac biomarkersof interest in the bodily fluid sample.
 2. The system of claim 1,wherein the bodily fluid sample is a blood sample.
 3. The system ofclaim 1, wherein the multiple cardiac biomarkers are selected fromC-reactive protein, Creatinine Kinase, TroponinT, Myoglobin, IL-6,IL-18, Brain Natriuretic Peptide, and D-Dimer.
 4. The system of claim 1,wherein the one or more aptamers are conjugated with biotin.
 5. Thesystem of claim 1, wherein the immobilization agent is selected from thegroup consisting of avidin, streptavidin, neutravidin and mixturesthereof.
 6. The system of claim 1, wherein the immobilization agent isapplied to a treating agent, and the treating agent is applied to themulti-array of vertically aligned platinum wires.
 7. The system of claim1, wherein the one or more aptamers are effective to impedimetricallydetect simultaneously the multiple cardiac biomarkers in the bodilyfluid sample, to provide a quantitative electrochemical impedanceread-out indicative of a presence or absence of the multiple cardiacbiomarkers of interest in the bodily fluid sample.
 8. The system ofclaim 1, wherein the multi-array of vertically aligned platinum wiresare arranged in a circular configuration.
 9. A method of simultaneouslydetecting multiple cardiac biomarkers in a bodily fluid sample of apatient, comprising: obtaining the bodily fluid sample from the patient;forming a detection device, comprising: forming a multi-arrayaptasensor, comprising: providing a conductive material interface,comprising: providing an epoxy substrate; obtaining a multi-array ofvertically aligned platinum wires; and casting the multi-array ofvertically aligned platinum wires in the epoxy substrate; polishing thesurface of the multi-array of vertically aligned platinum wires; forminga biological sensor agent, comprising: applying an immobilization agentto the multi-array of vertically aligned platinum wires; selecting oneor more aptamers to interact with the immobilization agent and toselectively bind with the multiple cardiac biomarkers of interest; andinteracting the multi-array aptasensor with the immobilization agent;contacting the multi-array aptasensor with the bodily fluid sample tosimultaneously detect multiple cardiac biomarkers of interest in apatient; generating an electrochemical impedance signal as a result ofthe multi-array aptasensor binding with the multiple cardiac biomarkersof interest; and assessing a presence or an absence of a change inelectrochemical impedance, wherein, the presence of a change in theelectrochemical impedance is indicative of a presence of the multiplecardiac biomarkers of interest in the bodily fluid sample and binding ofthe multi-array aptasensor with the multiple cardiac biomarkers in thebodily fluid, and wherein, the absence of a change in theelectrochemical impedance is indicative of an absence of the multiplecardiac biomarkers of interest in the bodily fluid sample.
 10. Themethod of claim 9, wherein the electrochemical impedance signal istransduced to a read-out value.
 11. The method of claim 10, wherein theelectrochemical impedance signal is connected to a portable, hand-helddevice that is effective to display the read-out value.
 12. The methodof claim 9, wherein the detection device is in the form of a test stripand the method, comprises: contacting the bodily fluid sample with thetest strip; assessing a visual change to the test strip; correlating thevisual change with a chart or key; and based on the correlation,determining if the visual change is indicative of the presence of achange in electrochemical impedance and the presence of the multiplecardiac biomarkers in the bodily fluid sample.
 13. The method of claim12, wherein the visual change is a color change.
 14. The method of claim9, wherein the polishing is conducted with a grit size in a range fromabout 320 grit to about 2400 grit.